Vitamin b12-active substance elution from montmorillonite absorbent



atented June 23, 1953 UNITED STATES TENT OFFICE VITAMIN Bra-ACTIVE SUBSTANCE ELUTION FROM MONTMORILLONI'IE ABSORBENT No Drawing. Original application September 7,

1950, Serial No. 183,664.

Divided and this application June 11, 1952, Serial No. 292,986

4 Claims. 1

This invention relates to an improved method for obtaining anti-pernicious anemia active substances from fermentation broth, and more particularly to a process for recovering vitamin B12 and vitamin Biz-like substances from montmorillonite adsorbents.

This application is a division of our copending application Serial No. 183,664, filed September 7, 1950.

It has been found previously that vitamin 1812 can be isolated from aqueous solutions by adsorbing the vitamin on a solid adsorbent such as activated charcoal or fullers earth, eluting the adsorbed vitamin B12 with an aqueous solution of an organic base, such as pyridine and the like, and recovering vitamin B12 from the resulting eluate. While this process is useful for isolating and recovering vitamin 1312, the use of pyridine and other organic bases in the eluting solution does have several serious disadvantages. One disadvantage is the serious loss of active principles which occurs during the processing. Further, the organic bases are expensive and it is therefore economically necessary to provide a means and equipment to recover these valuable chemicals. In addition, the use of eluting agents containing organic bases in recovering vitamin 3312 from montmorillonite adsorbates such as fullers earth is difhcult to carry out on a commercial scale, since the slurry of adsorbate and eluting agent is slimy and consequently extreme ly diincult to filter. Therefore, for most eificient operation, it was necessary to use activated charcoal as the adsorbing agent since the use of this adsorbent eliminated the operational dimculties previously encountered with fullers earth. However, the use of activated charcoal is uneconomical since it considerably more expensive than fullers earth, and the amount of charcoal required is about 4 or 5 times more than is required when fullers earth is used.

This invention is concerned with an improved method whereby vitamin B12 and vitamin B12 like substances may be recovered economically and in enhanced yields. It is one object of this invention to provide a practical process for recovering vitamin B12 and vitamin Biz-like substances from montmorillonite adsorbents such as iullers earth (calcium montmorillonite) or bentonite (sodium montmorillonite) utilizing only an aqueous inorganic solution for eluting these substances, and thereby avoiding the disadvantages of prior art procedures. It is a further object to provide a practical process for recovering and concentrating the vitamin and vitaminlike substances from solutions containing only minor amounts of said materials, utilizing the inexpensive montmorillonites as adsorbents and our improved eluting procedure. Other objects Will be apparent from the detailed description of our invention hereinafter provided.

The foregoing and other objects of this invention are achieved by eluting vitamin B12 and vitamin-like substances from montmorillonite adsorbents with an aqueous solution containing a minor amount of an anion of a nitrogen containing acid such as cyanide, thiocyanate or nitrite. W e have found that an aqueous solution containing these anions and having a pH of at least 6.0 Will effectively elute vitamin B12 and Biz-like substances from montmorillonite adsorbents. Our process, in addition to providing an inexpensive eluting agent, also permits the recovery of enhanced yields of these important products which are therapeutically active in treating pernicious anemias. Further, it permits the use of an inexpensive montmorillonite in place of carbon to adsorb the vitamin B12 and vitamin Biz-like substances from sources such as fermentation broths.

In accordance with one embodiment of our invention, our improved elution procedure is efiected by intimately contacting the montmorillonite adsorbent With the aqueous eluting solution for a time suificient to deadsorb the active principles. For example, this can be accomplished by stirring the eluting solution with the vitamin containing adsorbent and removing the spent adsorbent by filtration to recover an aqueous solution containing the desired active substances. The vitamin B12 and vitamin Biz-like substances contained in more concentrated form in the aqueous eluates of our invention are then readily processed further to recover these products in purified form for therapeutic or other use.

In carrying out the improved procedures of our invention, it is necessary that the eluting solution have a plT-l of about 6.0 or greater, since at lower pHs the active principles remain adsorbed to a. great extent and are not eluted by the solution. While, in general such aqueous solutions having a pH of at least 6 are useful in practicing our invention, We find that under optimum conditions best results are obtained by utilizing eluting solutions having a pH of about 'i' to 9.

The eluting solution is most conveniently prepared by dissolving a stable alkali metal, alkaline earth metal, or ammonium salt of the desired acid in water, and it necessary, adjusting the pH to the desired level by the addition of a weak base such as ammonium hydroxide or an alkali metal carbonate or bicarbonate. Frequently, because of the acidic nature of the vitamin adsorbate being eluted we find it desirable to add an excess of such a weak base in order to neutralize the acidity of the adsorbate and maintain the eluting solution at the optimum pH.

The quantity and concentration of eluting solutions used will, of course, depend upon the particular adsorbate being eluted. Usually, we find that about to parts by weight of the eluting solution per part of adsorbate is satis factory, although larger or smaller proportions of eluting solutions may be advantageous with certain adsorbates. While various concentrations of the inorganic salts can be used, we find that an amount of about 0.05 to 0.1% by weight of the inorganic salt in the eluting solution is usually sufiicient to permit the obtainment of anop'timum yield of the active principles.

The aqueous eluate obtained in accordance with the present invention may be processed. further to recover the pure vitamin B12 or vitamin Biz-like substances. The further treatment of the eluates will depend upon the anion used for the elution and the active principle to be recovered.

In carrying out the elution with a solution of a cyanide, the vitamin B12 and vitamin Bl2'1ike compounds contained on the adsorbent are converted to a purple colored cyanide complex. This cyanide complex is decomposed on treatment with an acid, resulting in the formation or vitamin B12. The vitamin 1312 may be recovered from the acidified solution by utilizing a purification procedure such as transferring the active substances from water to benzyl alcohol, chromatographing from activated alumina or charcoal, or distributing the active principle between benzyl alcohol and water in a multi-plate countercurrent distribution. Alternatively, two or more of these purification procedures may be used incombination.

When the. aqueous eluting solution employed contains thiooyanate or nitrite anions, the resul ing eluate, contains both, vitamin B 2 and vitamin Bra-like substances. These eluates can be further processedby the. purification methods indicated above to obtain vitamin B12 and/or the vitaminBm-like substances. However, generally We prefer to treat this mixture of vitamin B12 and vitamin Biz-like substances with a source of cyanide ions in order to convert the vitamin B12- like substances to vitamin B12. In this way it is possible to avoid the difficulty of separating the vitamin B12 and vitamin B12-like. substances and recover only oneproduct.

Pursuant to a further embodiment of ourv invention, we have found that vitamin. B12 and vitamin B12-like substances may be recovered in a more concentrated form from fermentation broths and the like which contain only minor. amounts of these principles. This is accomplished by adsorbing these minor amounts of vitamin andvitamin-like substances on a montmorillonite adsorbent, and eluting the active sub.- stances therefrom with an aqueous eluting solution such as those described previously. The eluates so obtained contain the active principles in considerably more concentrated form from which they may be recovered in purified form for therapeutic use by the methods previously indicated. Thus there is provided by our. invention a practical and economical method for. re.-

covering vitamin B12 and the vitamin B1z-like substances.

Our process is particularly useful in recovering vitamin B12 and vitamin Biz-like substances from spent streptomycin broths. Such spent broths are obtained, for example, by removing the streptomycin from the fermentation broth by adsorbing the antibiotic on a carboxylic acid type resin. To the resulting streptomycin-free broth is then added the montmorillonite adsorbent which removes the vitamin B12 and vitamin B12-like substances from the solution. The vitamin containing adsorbate is then separated from the spent broth as by filtration and further processed to recover the active principles in purified form as described above. For commercial operation we prefer to use fullers earth as the 1nontmorillonite adsorbent since this adsorbent is readily available and offers the minimum of processing difliculties. In actual practice, we find that about one part by Weight of fullers earth per 400 parts of broth are sufficient to completely adsorb the vitamin B12 and vitamin Eda-like substances.

The term vitamin B1zlike substances as used herein refers to those substances which like vitamin B12 are active in the treatment of pernicious anemias, and are converted to vitamin 1312 by reaction with a source of cyanide ions. They may be considered to be analogs of vitamin B12 wherein the cyanide group of this vitamin is replaced by other radicals.

The following examples are presented to illustrate specific embodiments of our invention:

Example I Fermentation broth obtained from the propagation of a streptomycin-producing strain of .S'. griscus and from which the streptomycin had been removed by adsorption on a carboxylic acid resin, such. as a granular copolymer of methacrylic acid and divinyl benzene containing 5% divinyl benzene, was acidified to pH 2.5. 0.10% by weight of fullers earth and an equal amount of diatomaceousearth filter aid were added. The mixture was stirred about 30 minutes and filtered.

To one portionoi the. adsorbate was added an aqueous solution containing 28% ammonium hydroxide and 0.1% sodium cyanide, 5 parts of solutionbeing employed per part of fullers earth. The mixture was. stirred about 30 minutes and filtered.

The resulting filtrate was then processed fur ther to recover and isolate the vitamin B12. The filtrate was first neutralized to pH '1 with acetic acid. and. to the neutral filtrate was added 50 g./liter. of sodium bicarbonate and 100 g./liter of sodium sulfate. The solution Was extracted once with volume and twice with volume of benzyl alcohol. The combined benzyl alcohol extracts of active substances were dried with anhydrous sodium sulfate. To the dried benzyl alcohol solution were added 5 volumes of acetone 'and;2 volumes of ether, to precipitate the active substances. The precipitate was removed by filtration and was dissolved in a minimum amount of methanol. The methanol solution chromatographed on activated alumina. The alumina column was eluted with methanol containing about 2% acetic acid, the fractions of red color were collected, andthe optical density was measured at- 5500 A. (characteristic maximum for vitamin B12). The measurement showed that 166 mg. of potential vitamin B12 (-98%. of.

Example 2 Fermentation broth of the type described in Example 1 and after acidification to pH 2.5 was treated with 0.10% by weight of fullers earth. One portion of the adsorbate was eluted with an aqueous solution containing 3% sodium bicarbonate and 0.05% sodium cyanide, said solution being employed in a ratio of 5 parts by weight per part of fullers earth. A second portion of the adsorbate was similarly eluted with an aqueous solution containing 1% sodium bicarbonate and 0.05% sodium cyanide.

The resulting eluates were then processed to recover the vitamin B12 as follows: 300 g./liter of ammonium sulfate was added, and the solution was extracted once with volume and twice with volume of benzyl alcohol. The combined benzyl alcohol extracts were acidified to pH 6-6.5 with acetic acid, filtered, and'dried over sodium sulfate. To the dried solution was added 60% by volume of carbon tetrachloride, and the solu tion was extracted four times with water, each extract being the volume of the zenyl alcohol. The active substances present in the water extracts were again transferred to benzyl alcohol, back to water, and back to benzyl alcohol, in the same manner. The benzyl alcohol solution was dried over anhydrous sodium sulfate, precipitated with 5 volumes of acetone and 2 volumes of ether, dissolved in a minimum amount of methanol, acidified to pH 4.0 with acetic acid, precipitated with 7 volumes of acetone and 2 volumes of ether, and dissolved in a minimum amount of methanol containing 0.1 ml. of glacial. acetic acid per 15 ml. of methanol. This solution was chromatographed on activated alumina. The alumina column was eluted with methanol, and the redcolored fractions were collected. The optical densities of these fractions were measured at 5500 ii. The measurements showed that 255 mg. and 21'? mg. of potential vitamin 1312 per 1000 gals. of broth. were present in the products derived from 3% and 1% sodium bicarbonate elu= tion, respectively.

Example 3 The procedure described in Example 2 was repeated employing an aqueous eluting solution containing 2% of sodium bicarbonate and 0.05% of potassium cyanide. Using the same method described in the preceding example, it was found that the resulting product contained 232 mg. of potential. vitamin B12 per 1000 gals. of broth.

When this experiment was repeated using an aqueous eluting solution containing only 2% of sodium bicarbonate, the resulting product was found to contain only 5 mg. of potential vitamin B12 per 1000 gals. of broth.

The results of these tests indicate that practically no active material is recovered when the eluting solution does not contain the cyanide ion.

Example 4 The procedure of Example 2 was repeated emd ploying three different solutions in the elution of fullers earth; (1) an aqueous solution containing 2% sodium bicarbonate and 0.1% sodium thiocyanate, '(2) an aqueous solution containing 2% sodium bicarbonate and 0.1% sodium nitrite, and (3) an aqueous solution containing 2% sodium bicarbonate, 0.1% sodium nitrite, and 2% so= dium chloride. Additionally, the first water extracts of 'benzyl alcohol after the fullers earth elutions were treated with 1 gm. of sodium cyanide per 400 ml. of solution (considerable excess) to convert vitamin B12'1ike substances and vitamin B12 to the vitamin Biz-cyanide complex. The amounts of potential vitamin B12 present in the products derived from the several eluting so=- lutions described above were (1) 192 mg. (2) 224 mg, and (3) 258 mg. per 1000 gals. of broth.

Bentonite, which also has a high adsorptive capacity for vitamin B12 and vitamin Biz-like substances, was used as the adsorbent to remove these active materials from solution. The adsorbate was eluted with solution (3) above, and the eluate was processed in the same general manner. The recovery of potential vitamin B12 corresponded to that obtained from fullers earth adsorbate.

Example 5 Crystalllzable Vitamin B12 mg./1,000 gels. of broth Batch No.

Average Various changes and modifications may be made in our invention, certain preferred embodiments of which are herein described, without departing from the scope thereof. It is our intention that such changes and modifications, to the extent that they are within the scope of the appended claims, will be construed as part of our invention.

We claim:

1. The process for recovering vitamin B12 active substances from a montmorillonite adsorbent which comprises eluting said vitamin B12 active substances with an aqueous solution having a pH in excess of about 6 and containing a source of nitrite ions.

2. The process as defined in claim 1 wherein the montmorillonite adsorbent is bentonite.

3. The process as defined in claim 1 wherein the montmorillonite adsorbent is fullers earth.

4. The process for eluting vitamin B12 active substances from a fullers earth adsorbate which comprises intimately contacting said adsorbate 7 8 with an aqueous solution having a pH of 7 to 9 Number Name Date and containing a source of nitrite ions. 2,563,794 Rickes Aug. 7, 1951 STEPHEN KUTOSH. GEORGE B. HUGHEY. FOREIGN PATENTS ROBERT s. MALCOLMSON. 5 Number Country Date 473,064 Great Britain Oct. 5, 1937 References Cited in the file of this patent OTHER REFERENCES UNITED STATES PATENTS Rickes: Science, volume 107, pages 396, 397 Number Name Date 10 (April 16, 1948).

2,134,256 Laland Oct. 25, 1938 Vitamins and Hormones, volume III (1945),

2,530,416 Wolf Nov. 21, 1950 pages 269, 270. 

1. THE PROCESS FOR RECOVERING VITAMIN B12 ACTIVE SUBSTANCES FROM A MONTMORILLONITE ADSORBENT WHICH COMPRISES ELUTING SAID VITAMIN B12 ACTIVE SUBSTANCES WITH AN AQUEOUS SOLUTION HAVING A PH IN EXCESS OF ABOUT 6 AND CONTAINING A SOURCE OF NITRITE IONS. 